Three-dimensional graphene/Pt nanoparticle …...Microbial fuel cells (MFCs) are able to directly convert about 50 to 90% of energy from oxidation of organic matters in waste to electricity

R E S EARCH ART I C L E

ENERGY RESOURCES

1State Key Laboratory of Urban Water Resource and Environment (Harbin Institute ofTechnology), Harbin 150080, China. 2School of Materials Science and Engineering, HarbinInstitute of Technology, Harbin 150080, China. 3CAS Key Laboratory of Nanosystem andHierarchical Fabrication, National Center for Nanoscience and Technology, Beijing 100190,China. 4Key Laboratory of Urban Pollutant Conversion, Institute of Urban Environment,Chinese Academy of Sciences, Xiamen 361021, China.*These authors contributed equally to this work.†Corresponding author. E-mail: [email protected] (S.L.); [email protected] (Z.T.)

Zhao et al. Sci. Adv. 2015;1:e1500372 13 November 2015

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10.1126/sciadv.1500372

Three-dimensional graphene/Pt nanoparticlecomposites as freestanding anode for enhancingperformance of microbial fuel cells
Shenlong Zhao,1,2,3* Yuchen Li,1* Huajie Yin,3 Zhouzhou Liu,1 Enxiao Luan,1 Feng Zhao,4
Zhiyong Tang,3† Shaoqin Liu1†

Microbial fuel cells (MFCs) are able to directly convert about 50 to 90% of energy from oxidation of organicmatters in waste to electricity and have great potential application in broad fields such as wastewater treat-ment. Unfortunately, the power density of the MFCs at present is significantly lower than the theoretical valuebecause of technical limitations including low bacteria loading capacity and difficult electron transfer betweenthe bacteria and the electrode. We reported a three-dimensional (3D) graphene aerogel (GA) decorated withplatinum nanoparticles (Pt NPs) as an efficient freestanding anode for MFCs. The 3D GA/Pt–based anode has acontinuous 3D macroporous structure that is favorable for microorganism immobilization and efficient electrolytetransport. Moreover, GA scaffold is homogenously decorated with Pt NPs to further enhance extracellular chargetransfer between the bacteria and the anode. The MFCs constructed with 3D GA/Pt–based anode generate a re-markable maximum power density of 1460 mW/m2, 5.3 times higher than that based on carbon cloth (273 mW/m2).It deserves to be stressed that 1460 mW/m2 obtained from the GA/Pt anode shows the superior performanceamong all the reported MFCs inoculated with Shewanella oneidensis MR-1. Moreover, as a demonstration of thereal application, the MFC equipped with the freestanding GA/Pt anode has been successfully applied in drivingtimer for the first time, which opens the avenue toward the real application of the MFCs.

INTRODUCTION

Treatment of organic wastewater currently needs both scientific andtechnical innovation because of huge energy costs; for instance, it con-sumes about 3 to 5% of all electrical power produced in the UnitedStates (1). Noteworthily, organic wastewater itself contains about ninetimes more energy than is needed for wastewater treatment (2). Thus,finding effective methods to use the energy stored in organic waste-water would not only decrease the energy consumption during waste-water treatment process but also offer novel routes to harvest the energy(3–5). Among different strategies, microbial fuel cells (MFCs), whichdirectly convert the chemical energy of organic matter such as carbo-hydrate and organic acids to electricity using microorganisms, havereceived specific attention because of their dual benefit: using waste-water or complex solid waste as fuel to produce electrical power as wellas reducing the biochemical oxygen demand loading of the wastewaterwith lower greenhouse emissions (6–9). Furthermore, in terms of en-ergy balance, MFCs are able to directly convert about 50 to 90% of en-ergy from oxidation of organic matters to electricity (10, 11), so intheory the amount of power recovered from wastewater by MFCs canhalve the electricity needed in whole wastewater treatment system (12).Actually, MFCs have demonstrated broad application prospects inwastewater treatment (13, 14), bioremediation (15), toxic metal recov-ery (16), marine sediment (17), desalination (18–20), and energy re-covery from human excrement in space (21). Unfortunately, the powerdensity of the MFCs at present is significantly lower than the theoretical

value (22–25). The observed low power should originate from currenttechnical limitations such as low bacteria loading capacity and the dif-ficult electron transfer between the bacteria and the electrode.

Among all the factors that affect the performance of MFCs, theselection and construction of anode materials are critical, which gov-ern overall performance of MFCs (26–29). First, an ideal anode ofMFCs that provides the accommodation space of bacteria requires po-rous structure and good biocompatibility, ensuring effectiveimmobilization, growth and cultivation of living microorganisms, fac-ile substrate transport to attached microorganisms, and easy re-moval of waste products. Second, the anode should be an excellentconductor to facilitate the electron transfer released from the micro-organisms and efficient collection of currents from all regions of theelectrode (30–32). Finally, for the real application of MFCs, an optimalanode needs high chemical and physical stabilities. A number ofmaterials have been examined as the MFC anodes including noblemetals (23–25), carbonaceous electrodes of distinct packing modes (forexample, carbon cloth, graphite fiber brushes, carbon felt, carbon mesh,and carbon paper) (33–39), and conductive polymers and their com-posites (40–44). Although these anode materials bring considerableincrement in the electric current, they are also reported to have someinherent drawbacks. For example, high cost and weak adhesion of in-oculated bacteria prevent practical use of noble metal electrodes inMFCs. Although many features of the carbonaceous electrodes includ-ing the high mechanical strength, enhanced conductivity, eco-friendlycomposition, and cheap cost satisfy most of the requirements of MFCelectrodes, they are generally nonporous and have poor electrical con-ductivity and low extracellular electron transfer efficiency compared tonoble metals. The carbon-based anodes are hybridized with carbonnanotubes (45–48), graphene (28, 49, 50), noble metal nanoparticles(51, 52), and conductive polymers (53, 54) to improve bacteria loadingcapacity and promote the direct electron transfer. Modification of the

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anodes results in enhancement of the electrode surface area, reductionof the internal resistance, and thereby increase of the MFC efficiencies.Nevertheless, novel material and structure design about the anodes arehighly desirable to further improve MFCs performance and life dura-bility for practical application.

Here, we reported three-dimensional (3D) graphene aerogel (GA)decorated with platinum nanoparticles (Pt NPs) as an efficient free-standing anode for MFCs. The proposed GA-based anode has a con-tinuous 3D macroporous structure that is favorable for microorganismimmobilization and efficient electrolyte transport. Moreover, to fur-ther enhance extracellular charge transfer between the bacteria andthe anode, GA scaffold is homogenously decorated with Pt NPs. Thecharge transfer capability of Pt NPs benefits the transfer and collectionof the generated electrons to the anode (51, 52). As a result, the MFCsconstructed with the 3D GA/Pt–based anode generate an exceptionalhigh current density of 4.88 A/m2 and power density of 1460 mW/m2,and the possible mechanism involved is also discussed. Impressively,as a demonstration of the real application, the MFCs constructed withthe GA/Pt anode successfully run a timer for the first time, whichopens the avenue toward the real application of the MFCs.

RESULTS AND DISCUSSION

To prepare the 3D GA/Pt–based anode, we synthesized self-supported3D GA according to the previously reported hydrothermal treatmentof graphene oxide (GO) sheets (55, 56). Raman spectrum survey re-veals that GO in the product is reduced, and its conjugated structuresare partly restored (fig. S1). Subsequently, 3D GA was decorated with


Pt NPs using a facile and surfactant-free microwave-assisted polyolprocess (see Materials and Methods). The obtained GA/Pt exhibitsremarkable mechanical property including high strength and excellenttoughness and can be arbitrarily tailored into any shapes, such as cubes,cylinders, or strips to fit different device configurations (Fig. 1A). Themicroscopic structure of GA/Pt is presented in Fig. 1B. It is noted thatnumerous flexible graphene sheets cross over each other to form char-acteristic 3D open porous architectures. Higher magnification (Fig. 1C)reveals that the pores with sizes ranging from several micrometer totens of micrometer are interconnected, which are large enough to al-low for internal colonization of Shewanella oneidensis MR-1 [the sizeof S. oneidensisMR-1 is about 0.5 mm × 2.5 mm (36)] as well as efficientelectrolyte transport. Transmission electron microscopy (TEM) images(Fig. 1, D and E) further disclose that the Pt NPs with monodispersesizes of 2.5 ± 0.3 nm are uniformly distributed on the surface of graphenesheets. The loading amount of Pt NPs has an important influence onelectronic conductivity, roughness, and hydrophilic property of GA; hence,it would affect the inoculation of bacteria cells onto the surface ofthe electrode, sustainability of the bacteria cells in the the electrodeenvironment, as well as electrode transfer rate and durability (52, 57–60).So, the composition of the GA/Pt is analyzed by the energy dispersivespectroscopy (EDS) and the inductively coupled plasma–mass spectra(ICP-MS). EDS analysis confirms that the products are composed ofC(93.20 atomic %), O (5.60 atomic %), and Pt (1.20 atomic %) (Fig. 1F).ICP-MS result shows that the loading of Pt is about 16.61 wt % in GA/Pt,which is very close to that obtained by the EDS measurement.

To examine the conductivity and diffusion of as-synthesized anodematerials, we used electrochemical impedance spectroscopy (EIS) to

Fig. 1. The physical characterization of GA/Pt. (A) Digital photos of GA/Pt. (B and C) SEM and magnified SEM images of GA/Pt. (D and E) TEM and high-resolution TEM images of GA/Pt. (E) Inset: size distribution of Pt. (F) Energy-dispersive x-ray spectrum of GA/Pt, and the inset summarizes the content of
C, O, and Pt elements. a.u., arbitrary units.
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study the electrochemical behavior of GA and GA/Pt along with widelyused carbon cloth anode. The results are plotted as Nyquist curvesand further fitted with an equivalent circuit (Fig. 2 and the inset). EachEIS curve is composed of a well-defined semicircle followed by a straightline. The intercept of the semicircle with the real impedance axis presentsthe total ohmic resistance (Rohm) of the electrochemical cell includingthe solution resistance (Rs) and the charge transfer resistance (Rct) atthe electrode/electrolyte interface (61). The Rohm of GA/Pt anode is es-timated to be ~68.7 ohms (Fig. 2), much lower than that of carbon clothmaterials (~2790.6 ohms) and pure GA (~102.7 ohms). Moreover, theRct at the electrode/electrolyte interface, corresponding to the diameterof the semicircles, follows the order of carbon cloth (~2729.2 ohms) >GA (~42.9 ohms) > GA/Pt (~8.2 ohms). Because all three electrodes sharethe same setup and the same electrolyte, the decrease in the Rct from~42.9 ohms for GA to ~8.2 ohms for GA/Pt can only be attributed tothe presence of Pt NPs. This result highlights the effectiveness of PtNPs in enhancing charge transfer ability of GA. Except for Rohm andRct, the ion diffusion resistance is also an important parameter to con-tribute to the total internal resistance, which causes the power or en-ergy losses in MFCs (62, 63). The ion diffusion coefficient (D) can becalculated from the plots in the low-frequency region (the straight lineregion in Fig. 2) based on Eqs. (1) and (2) (64, 65)

Z′ ¼ Rs þ Rct þ sww−0:5 ð1Þ

D ¼ R2T2=ð2S2F4s2wC2Þ ð2Þ

where Z′ is the real part of the impedance, w is the angular frequency,and sw represents the slope of Z′ against w−0.5. R, F, T, S, and C are gasconstant, Faraday constant, absolute temperature, surface area, andmolar concentration of electrolyte ions, respectively. Thus, the D ofGA/Pt, GA, and carbon cloth is estimated to be 1.45 × 10−7 cm2/s,4.62 × 10−8 cm2/s, and 3.31 × 10−10 cm2/s, respectively. Notably, onecan see the two-order-of-magnitude improvement of D values ofGA/Pt and GA over carbon cloth, which should be attributed to easyion diffusion in 3D porous structure of GA/Pt and GA. Together, theEIS analysis reveals that GA/Pt composites with small charge transferresistance and good ion diffusion coefficient would be ideal anodematerials for high-performance MFCs.


To further evaluate the bacteria loading capacity and biocompati-bility of the 3D GA/Pt composites, after incubation with S. oneidensisMR-1 for 3 days, we cut both GA/Pt and GA samples and we exam-ined their interior. The scanning electron microscopy (SEM) images ofGA/Pt (fig. S2 and Fig. 3A) clearly show that the entire surface of GA/Ptis covered with rod-shaped bacteria cells. The magnified SEM imagesof the cross section of GA/Pt (Fig. 3B and fig. S2, B and C) and GA(fig. S3) reveal that the interior surfaces of GA/Pt and GA are also fullycovered with rod-shaped bacteria cells without clogging the macro-pores. These observations prove that the open macroporous structureof GA/Pt and GA has high bacteria loading capacity and allows forsufficient substrate transport to maintain internal colonization. As forthe commercial carbon cloth, the bacteria biofilms are formed onlyon the surfaces or cracks (Fig. 3C), which are caused by a relativelysmooth surface, small specific surface area, and low porosity of thecarbon cloth. The good biocompatibility of the GA/Pt composites isfurther confirmed with the stained assay with calcein AM and PI todistinguish the live (green) and dead/apoptotic cells (red), respectively(see the detailed explanation in Materials and Methods). Figure 3Dpresents the CLSM image of bacteria immobilized on the interiorsurfaces of GA/Pt. It can be found that almost no death is observedin the bacteria cells, indicating the good biocompatibility of GA/Ptcomposites (Fig. 3D).

The outstanding charge transfer ability and good biocompatibilitywith macroporous 3D structure of GA/Pt satisfies the ideal MFC anodeconfiguration. We then constructed the conventional H-shaped two-chamber MFCs by directly adopting the synthesized GA/Pt compositesas freestanding anodes. The GA/Pt electrode inoculated with S. oneidensisMR-1 was positioned in the anodic chamber fed with M9 buffer solu-tion containing 18mM sodium lactate. The cathode was Pt sheet electrode(1 cm × 1 cm), and the cathodic chamber was fed with potassiumhexacyanoferrate (K3[Fe(CN)6], 50 mM) and potassium chloride (KCl,50 mM) solutions. The uncolonized GA/Pt anode was initially inactive(Fig. 4A). After microbial inoculation with S. oneidensisMR-1 for 8 days,the open circuit voltage of MFCs increased to more than 0.3 V acrossa 1-kilohm resistor, indicating a successful startup (45). When theMFCs achieved stable power generation, its long-term stability was in-vestigated. The anode chamber was operated in fed-batch mode, and

Fig. 2. Conductivity measurement of GA/Pt. (A) Nyquist plots of GA, GA/Pt, and carbon cloth. The ac impedance spectra are fitted with an equivalentcircuit (inset), where Rs is the ohmic resistance of the electrolyte. Cd reflects the interfacial capacitance. Rct and RW are the charge transfer resistance of the
anode material and the Warburg impedance of ion diffusion in the electrode, respectively. (B) Amplified Nyquist plots of the three samples. Comparedwith the GA and carbon cloth, Nyquist plot analysis indicates that the GA/Pt anode with small charge transfer resistance and good ion diffusion coefficientwould be an ideal anode material for high-performance MFCs.
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the fresh medium was added when the current decreased to <0.5 mA.Figure 4B displays the typical power generation profile. The powerdensity of the MFC with GA/Pt anode reaches 1193 mW/m2 imme-diately after lactate injection and sharply decreases after running forabout 100 hours because of depletion of sodium lactate. After refresh-ing the culture medium, the power density of the MFC quickly recoversto the initiated value of 1197 mW/m2. Repeatable power generationcycles demonstrate the excellent stability of GA/Pt anode for electricityharvesting. The SEM image of the GA/Pt anode after 25 days of op-eration (three power generation cycles) indicates that the bacteria cellsfirmly adhere to the macroporous architecture of the GA/Pt and forma thick biofilm but do not clog the macroscale pores (Fig. 4C). More-over, a great number of microbial nanowires were observed underSEM imaging. These nanowires tethered cells to the anode surfaceor to other cells, providing a probable direct path for extracellular elec-tron transfer (66–69).

The long-term stability of the MFC with different substrate con-centration has also been investigated. It is found that varied substrateconcentration does not change the power density of MFCs; however,increasing the concentration of sodium lactate from 18 mM to 36 or180 mM can result in longer running time (fig. S4). For example, thepower density of the MFC with GA/Pt anode in 36 mM sodium lactatestarts to decrease after running for about 200 hours, whereas the elec-


trodes with abundant sodium lactate (180 mM) can be operated formore than 750 hours (ca. 31 days). The above results demonstrate thatthe decrease in the power density of the MFC with GA/Pt anode isascribed to depletion of sodium lactate, which is the only electron do-nor source for the S. oneidensisMR-1 in the MFC system. That is, thecontent of electron donor source is the dominating factor responsiblefor the degradation of the MFC under the ideal condition (70). Asdemonstrated in fig. S4, the MFC with GA/Pt anode still keeps theinitiated value of 1198 mW/m2 after 43 days of operation, highlight-ing the excellent stability of GA/Pt anode for electricity harvesting.Such high durability of GA/Pt anode would greatly benefit the practicalapplication.

The alive-dead stained assay further reveals that almost all micro-organisms remain alive (Fig. 4D), strongly suggesting that the goodbiocompatibility of the 3D macroporous architecture is responsiblefor the excellent stability of GA/Pt anode for electricity harvesting.For comparison, we also investigated the electricity harvesting efficien-cy of GA- and carbon cloth–based MFCs under identical conditions(fig. S5). As shown in fig. S5, although both the GA and commercialcarbon cloth anode could produce power, the current density of MFCsconstructed with GA/Pt anodes (2372 mA/m2) is 1.6 and 6.0 timeshigher than that of the GA anode (1481 mA/m2) and the commercialcarbon cloth anode (395 mA/m2). The coulomb efficiency of GA/Pt,GA, and carbon cloth anode is estimated to be 69.3, 42.6, and 8.1%,respectively (see the detailed calculation process in the SupplementaryMaterials), so it is evident that the electricity conversion efficiency ofGA/Pt anode is higher than that of GA and carbon cloth. The superiorperformance of GA/Pt should originate from good biocompatibilityand high electron transfer efficiency between the bacteria and theGA/Pt anode (see the possible mechanism and detailed descriptionin fig. S6).

The power density and polarization curves of three anodes (GA/Pt,GA, and carbon cloth) were determined by varying the external loadresistance (0.01 to 99999 ohms). As demonstrated in Fig. 5A, the MFCequipped with GA/Pt anode delivers a remarkable maximum powerdensity of 1460 mW/m2, 5.3 times higher than that based on carbon cloth(273mW/m2) and 1.8 times higher than that based on GA (811mW/m2).Moreover, the maximum current density of the GA/Pt anode (4.88 A/m2)is 5.5 times higher than that of the carbon cloth anode (0.88 A/m2).These data strongly suggest that the GA/Pt anode enables the superiorperformance among all the reported MFCs inoculated with S. oneidensisMR-1 (table S1). Furthermore, the GA/Pt composites can be easily madein any size (as shown in Fig. 1A), which makes them ideal as anodesfor small- or large-scale MFC applications. To exemplify the real ap-plication of the MFCs, two single biofuel cells have been assembled inseries and successfully run a timer (Fig. 5B and movie S1). To the bestof our knowledge, this is the first report to show that MFCs coulddrive the electrical devices.

Finally, we preliminarily examine the power generation capabil-ity of the MFC fed with wastewater. The effluent from the primarysettling tanks in a municipal wastewater treatment facility (GaobeidianWastewater Treatment Plant, Beijing) is directly used as fuels withoutany pretreatment (fig. S7). Impressively, the maximum current and powerdensity of the MFC fed with primary effluent can be up to 3.1 A/m2 and1064 mW/m2, respectviely, verifying the potential in the practicalwastewater treatment. Future work will be focused on rationalizationof 3D GA/Pt architecture (such as their longevity in wastewater, reactordesign, and reaction temperature) for large-scale MFC applications.

Fig. 3. The bacteria loading capacity and biocompatibility of GA/Pt.(A) SEM image of the surface of GA/Pt incubation with S. oneidensis
MR-1 (inset: magnified SEM image). (B) SEM image of the cross sectionof GA/Pt incubation with S. oneidensis MR-1 (inset: magnified SEM image),revealing that the open macroporous structure of GA/Pt has high bacterialoading capacity. (C) SEM image of carbon cloth anode incubation withS. oneidensis MR-1 (inset: magnified SEM image). By comparing with thetraditional carbon cloth under the same culture conditions, the advantageof GA/Pt for bacteria loading capacity is very obvious. (D) Confocal laserscanning microscope (CLSM) image of bacteria on GA/Pt after 3 days ofcolonization. The biocompatibility of GA/Pt composites is confirmed withthe stained assay with calcein AM and propidium iodide (PI) to distinguishthe live (green) and dead (red), respectively. CLSM image shows thatbacteria can live very well on the GA/Pt.
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CONCLUSIONS

In summary, we propose the 3D porous GA decorated with Pt NPs asa novel and high-performance anode of MFCs. The MFCs equippedwith the GA/Pt anode reach a current density of 4.88 A/m2 and gen-erate a maximum power density of 1460 mW/m2 in sodium lactate me-dium, much higher than that obtained from the carbon cloth or pureGA under the same conditions. Detailed structural and electrochemicalcharacterizations elucidate that the excellent performance of the GA/Ptanode is attributed to its high bacteria loading capacity, easy extracellularelectron transfer between the cytochrome protein of the bacteria and theGA/Pt, as well as fast ion diffusion in 3D pores. Notably, we demonstratethat the MFCs constructed with the GA/Pt anode successfully run a


timer. This is the first reportedMFC in the literature to run an electricaldevice, which opens the avenue toward the real application of the MFCs.

MATERIALS AND METHODS

MaterialsGraphite powder (natural briquetting grade of 99.999% purity) andH2PtCl6⋅6H2O were purchased from Alfa Aesar. Ultrapure water(Millipore Milli-Q grade) with a resistivity of 18.2 megohms was usedin all the experiments. GO was prepared from the graphite powderaccording to Hummers’ method.

Fig. 5. The single-cell performance testing of MFCs and its application. (A) MFC single-cell performance constructed with different electrodematerials working at room temperature. The current density (A/m2) was obtained by using the measured current (three times) and the electrode area.
(B) Digital photo of MFCs to drive a timer. The two single biofuel cells have been assembled in series and successfully run a timer, strongly exemplifyingthat the GA/Pt anode enables the superior performance and the actual application potential.
Fig. 4. The stability and repeatability of MFC constructed with GA/Pt. (A) Voltage generation of MFC across a 1-kilohm external resistor, and anoperating voltage higher than 0.3 V indicates successful startup. (B) Repeatable power generation cycles with a 1-kilohm loading. Red and pink arrows
point to sodium lactate feeding and refresh. (C) SEM image of bacteria on GA/Pt anode after 25 days of operation. The bacteria cells firmly adhere to themacroporous architecture of the GA/Pt and form a thick biofilm after 25 days of operation. (D) CLSM image of bacteria on GA/Pt anode after 25 days ofcolonization. After 25 days of operation, almost all of the bacteria are live (green) and few are dead because of the normal apoptotic cells.
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Synthesis of 3D architectures of GAFirst, 60 ml of GO aqueous solution (2 mg/ml) was mixed with 100 mlof NH3⋅H2O under mild ultrasonication. The mixture solution wasthen heated at 180°C for 12 hours to form GA. After the productwas dialyzed with ultrapure water for at least 1 day, it was subjectedto freeze-drying to generate 3D GA.

Synthesis 3D architectures of GA/Pt anode3D GA (80 mg) was dispersed in 60 ml of ethylene glycol followed byaddition of 1.5 ml of 80 mM H2PtCl6. After the mixture solution wasstirred for 1 hour, its pH value was adjusted to 11 by adding 1MNaOHsolution. After the mixture was gently stirred for another 12 hours, itwas placed in a microwave oven (CEM Voyager, 800 W) and heatedby microwave irradiation for 60 s. Finally, the resulting GA/Pt anodewas taken out, washed with deionized water, and dried in a vacuumoven at 60°C for 12 hours.

Physical characterizationTEM imaging was performed on an FEI Tecnai G2 F20 electron mi-croscope operated at 200 kV. The surface morphology was observedwith a Hitachi S4800 SEM. The ICP-MS were conducted with NEXION300X. The GA/Pt after incubation with bacteria was fixed by immersionin 2.5% glutaraldehyde solution [5 ml of 50% glutaraldehyde, 45 mlof water, 45 ml of 0.2 mM phosphate-buffered saline (PBS) buffer]overnight at 4°C and chemically dehydrated using ethanol (30, 50, 70,85, and 95% for one time, 100% for two times) with gradient concen-tration. The sample was dried in air and finally sputter-coated with10-nm gold for SEM imaging. The EIS was recorded in PBS buffer(pH 7.02) using CHI 660D electrochemical working station (Chen Hua,Shanghai, China) with saturated calomel electrodes as a reference elec-trode. EIS was measured at the open circuit voltage in the frequencyrange of 105 to 0.05 Hz with a 10-mV peak-to-peak sinusoidal poten-tial perturbation. The results were reported as Nyquist plots.

Bacteria cultureS. oneidensis MR-1 was purchased from the American Type CultureCollection (ATCC 700550) and stored at −80°C before use. S. oneidensisMR-1 was first grown in NB medium [Nutrient Broth, Bacto Tryptone(10 g/liter), beef extract (3 g/liter), NaCl (5 g/liter)] for 12 hours andthen streaked ontoNA [Nutrient Agar, Bacto Tryptone (10 g/liter), beefextract (3 g/liter), NaCl (5 g/liter), agar (15 g/liter)] plates. The resultingcolonieswere inoculated in 200ml of freshNBand incubatedwith shakingat 37°C until the OD600 (optical density at 600 nm) reached about 1.0.

MFC experimentH-shaped two-chamber MFC, which was constructed by connectingtwo 50-ml L-shaped media tubes 2.5 cm in diameter and separated byNafion membrane, was used in this work. The cathode was Pt sheetelectrode (1 cm × 1 cm), and the cathodic chamber was fed with po-tassium hexacyanoferrate (K3[Fe(CN)6], 50 mM) and potassium chlo-ride (KCl, 50 mM) solutions. The anode was blank or preinoculatedGA/Pt (1.5 cm × 1.5 cm), GA (1.5 cm × 1.5 cm), or carbon cloth (1.5 cm× 3.0 cm). In the case of the blank anode, the 5-ml bacteria solution,in which OD600 reached 1.0, was pelleted by centrifugation, washedwith M9 buffer [10 mM succinic acid, 5.7 mM Na2HPO4, 3.3 mMKH2PO4, 18 mM NH4Cl, 1 mMMgSO4, and L-cysteine (0.02 g/liter)],and resuspended in 45 ml of M9 buffer supplemented with 18 mMlactate. The NB medium (5 ml) was then added to the above buffer


solution and purged with pure nitrogen gas for 30 min to remove thedissolved oxygen. The anodic chamber was tightly sealed to maintainthe anaerobic condition during the MFC operation. In the case of pre-inoculated anode, the sterile GA/Pt anode was first inoculated in200 ml of fresh NB with the resulting colonies on the NA plates. WhenOD600 of bacteria reached about 1.0, the preinoculated GA/Pt wasmoved to the anode chamber of MFC for electricity harvesting. Theelectrolyte was 45 ml of M9 buffer containing 18 mM sodium lactateand 5 ml of NB solution. All the MFCs were operated on laboratorybench tops at ambient temperatures (~25°C). The anode and the cath-ode were connected through a variable external resistance of 0.01 to99999 ohms, and the voltage across the resistor was monitored withdigital multimeter (DT 9205A+). At the steady state of MFC, thepolarization curves were obtained by varying the external resistor. Themeasured potential was converted to an electric current using the fol-lowing equation: I = V (output voltage)/R (external resistance). Wecalculated the current density (A/m2) using the measured current andthe electrode area. The power density (P) (W/m2) was obtained bythe following equation: P = V × I.

CLSM measurementThe live-dead assay was used to evaluate the bacteria viability on theGA/Pt composite. The GA/Pt after incubation with bacteria for differ-ent times was crushed into small pieces, rinsed with PBS, and stainedwith calcein AM (Invitrogen) and PI (Invitrogen), respectively. The stainedcells were examined using a Leica TCS SP5 II confocal/multiphotonlaser scanning microscope (Germany). The live-dead assay solutionwas prepared by diluting calcein AM (1 mM solution in dimethylsulfoxide) and PI (1.5 mM solution in deionized water) stock solutionsin PBS at final concentrations of 2.0 and 4.0 mM. Calcein AM is a cell-permeant and nonfluorescent compound that was widely used for de-termining cell viability. In live cells, the nonfluorescent calcein AMwas hydrolyzed by intracellular esterases into the strongly green fluo-rescent anion calcein. The fluorescent calcein was well retained in thecytoplasm in live cells. PI, a red fluorescence dye (excitation/emissionmaxima of ~535/617 nm when bound to DNA), was used to differ-entiate necrotic, apoptotic, and normal cells.

SUPPLEMENTARY MATERIALSSupplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/10/e1500372/DC1Fig. S1. Raman spectra of GO and GA.Fig. S2. SEM images of GA/Pt incubated with S. oneidensis MR-1.Fig. S3. SEM images of bacteria on GA with different magnifications.Fig. S4. Long stability power generation profile of MFC by adding 18, 36, or 180 mM sodiumlactate in fed-batch mode.Fig. S5. Power generation profiles of MFCs constructed with carbon cloth (dark), GA (green),and GA/Pt anodes (red).Fig. S6. Illustration of possiblemechanism involvingGA/Pt anode for enhancing theMFCperformance.Fig. S7. MFC single-cell performance fed with primary effluent.Table S1. Summary of the performance of the previously reported MFCs inoculated with S.oneidensis MR-1.Movie S1. The real application of the MFCs for running a timer.References (71–81)

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Acknowledgments:We thank the Center for the use of FEI Tecnai G2 F20 electron microscopeand Hitachi S4800 SEM. Funding: We thank the Center for the use of FEI Tecnai G2 F20 electronmicroscope and Hitachi S4800 SEM. This work was financially supported by the National ResearchFund for Fundamental Key Project (grant 2014CB931801), National Natural Science Foundation ofChina (grants 51372054, 21161120325, 21025310, and 51402069), and Outstanding Young Fundingof Heilongjiang Province and State Key Laboratory of Urban Water Resource and Environment (HarbinInstitute of Technology) (grant 2015TS03). Author contributions: S.L. and Z.T. proposed the researchdirection and guided the project. S.Z. and Y.L. designed and performed the experiments. Y.L.separated and purified S. oneidensis MR-1. S.Z., Y.L., and H.Y. analyzed and discussed the ex-perimental results. S.Z., Y.L., S.L., and Z.T. wrote the manuscript. H.Y., Z.L., E.L., S.L., and F.Z.joined the discussion of data and gave some useful suggestions. Competing interests: Theauthors declare that they have no competing interests. Data and materials availability: All dataneeded to evaluate the conclusions in the paper are present in the paper and/or the SupplementaryMaterials. Additional data related to this paper may be requested from the authors.

Submitted 23 March 2015Accepted 1 September 2015Published 13 November 201510.1126/sciadv.1500372

Citation: S. Zhao, Y. Li, H. Yin, Z. Liu, E. Luan, F. Zhao, Z. Tang, S. Liu, Three-dimensionalgraphene/Pt nanoparticle composites as freestanding anode for enhancing performance ofmicrobial fuel cells. Sci. Adv. 1, e1500372 (2015).

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